The present invention relates to cell lines and transgenic animals. More particularly it relates to a method for producing a rat cell line, a method for producing a transgenic rat, a transgenic rat, a rat cell line, cells and tissue obtained therefrom and uses therefore.
Currently there are few permanently growing or immortalised cell lines of epithelial or neuronal origin which are fully characteristic of the differentiated phenotype or produce differentiated products at the levels seen in viva. For example, there are immortalised cell lines of human and rodent origin from the breast but these fail to express many of the characteristics of fully differentiated cells in vivo (Cancer Metastasis Rev. 6. 55-83, 1987 and Histol, Histopathol. 8 385-404, 1993). Those of the Central Nervous System (CNS) have limited proliferation capabilities whilst those that do proliferate are almost exclusively derived from neuroblastomas of peripheral nerves and are not representative of the CNS.
The present in vitro methods for examining differentiated epithelial and neuronal cell behaviour involves repeated establishment of primary cell cultures from rodent fetal tissues. Unfortunately these cultures rapidly senesce, contain mixed populations of cells and invariably are overgrown by contaminating, more rapidly growing cell types such as fibroblasts (J. Cell Biol. 73. 561-577, 1977 and In Vitro, 25, 23-36, 1989). They are therefore unsuitable for most molecular analyses where total cell extracts are required such as, for example, assays for messenger RNA, specific proteins, ligand-receptor interactions, and are wasteful in time and animals used for their production. The ability to produce immortalised populations of such cells would therefore be an enormous advantage for basic and applied biological studies. Since the rat is the laboratory animal used in most pharmacological studies, it is the animal of choice for any experimentally-derived cell systems for the pharmaceutical and toxicological industries.
Unfortunately the very process of immortalisation or transformation mitigates against the expression of the differentiated phenotype, since biological systems are almost invariably programmed only to differentiate after proliferation has ceased. Thus primary cultured cells, if they can be induced to proliferate, may be immortalised by chemical agents or more specifically by transforming or immortalising genes carried by oncogenic viruses (J. Virol 15., 613-618, 1985) eg. breast cells. (Dev. Biol. 136 167-180, 1989), but the resultant cell lines fail to express the levels of differentiated products or structures seen in vivo (Histol, Histopathol. 8, 385-404, 1993, J. Cell Biol. 91., 827-836, 1981, Eur.J. Biochem. 133, 707-715, 1983, J. Natl. Cancer Inst. 76, 246-256, 1986). If the primary cultures do not possess much ability to proliferate (eg. neuronal cells) then introduction of immortalising genes, by whatever route, fails to immortalise the cells, since several rounds of replication of the DNA are required to integrate successfully any transfected DNA into that of the host (J. Virol 15, 613-618, 1975).
There are essentially two approaches to the generation of cell lines.
1) One can isolate primary tissue cells from the tissue of interest and culture these in vitro. One can then attempt to immortalise these cells in vitro using various techniques before they senesce, using microinjection or other transfection techniques.
2) One can attempt to generate a transgenic animal from which to derive cell lines.
Primary Cell Transfection:
The most common way of immortalising primary tissue cells is to transform these with an immortalising gene construct. Such genes are commonly found in a variety of viruses. There are various problems with this technique. One needs a large number of primary cells as transformation is inefficient. Transformation will result in integration of the transforming construct into arbitrary sites in the genomic DNA. The genomic environment of the integration determines its levels of expression and experience shows that this tends to be highly variable so transformations of the same primary tissue often give rise to cell lines with widely differing properties. The immortalisation of cells also tends to suppress terminal differentiation which is disadvantageous if the cell line is supposed to mimic the behaviour of its tissue of origin.
One can use a conditional immortalising gene construct. An example is a temperature sensitive mutant, tsA58 (P. Tegtmeyer, J. Virol. 15, 613-618, 1975) which is found in the early region of the Simian Virus 40, and which encodes a thermolabile protein, the Large Tumour (LT) antigen which is capable of immortalising cells only at its permissive temperature (C. A. Petit, M. Gardes and J. Feunteun, Virology 127, 74-82, 1983, P. S. Cat and P. A. Sharp, Mol. Cell. Biol. 9, 1672-1681, 1989). This allows transfected primary tissue cells to be grown indefinitely in culture at the permissive temperature but if cells are required for experimentation then they can be cultured at the restrictive temperature and one would hope that they express genes in the manner of their primary tissue of origin. This does not overcome the problem of multiple and heterologous integration sites of one""s transformation constructs.
Cell Lines from Transgenic Animals:
Construction of a transgenic animal results in an organism that has an engineered construct present in all cells in the same genomic integration site. Thus cell lines derived from a transgenic animal will be consistent in as much as the engineered construct will be in the same genomic integration site in all cells and hence will suffer the same position effect variation. This is a small improvement over primary cell immortalisation.
(i) Tumour Derived Cell Lines:
The first and simplest approach has been to transfect cultured murine embryonic stem cells with an immortalising gene construct under the control of a cell type specific promoter. Transformed ES cells can then be injected into a blastocyst from a host mother and the host embryo reimplanted into the mother. One hopes to get a chimeric mouse whose tissues are composed of cells derived from types of embryonic stem cell present in the embryo. Usually the mice from which the ES cells for transformation are derived are chosen to have a different coat colour from the host mouse into whose embryos the transformed cells are to be integrated. Chimeric mice will then have a variegated coat colour. Such mice are then crossed with an appropriate strain in the hope that the germline will also be chimeric and that offspring mice will carry the transgene. It is then hoped that the transgenic mice will develop tumours in the tissues in which the promoter is activated. These tumours can then be cultured as cell lines.
It has been shown that mice transfected with a construct of the SV40 LT antigen under the control of the metallothionein promoter developed tumours of the choroid plexus, and that cell lines can be isolated from these transformed tissues (R. L. Brinster et al, Cell 37, 367-379, 1984). Similarly, it has been shown that in mice transfected with an LT-antigen construct under the control of the 5xe2x80x2 regulatory sequences of the insulin gene, tumours developed in the beta-islets of the pancreas (D. Hanahan, Nature 315, 115-122, 1985). This is a clear example of immortalising gene being targeted to the tissue of interest.
Despite these and other successes with this approach it is not ideal. Tumour formation is associated with multiple genetic abnormalities and chromosomal rearrangements (B. Vogelstein et al, N. Engl. J. Med. 319, 525-532, 1988). More often than not the resultant cells no longer express the relevant terminally differentiated genes, or at least not appropriately.
(ii) Conditional Immortalisation of Cell Tissues
A second approach is to transform one""s ES cells with a conditional immortalising gene coupled to a broad specificity promoter so that ostensibly the construct is expressed in all tissues in the mouse. If a temperature sensitive immortalising gene is used then the construct will be inactive at the body temperature of the mouse but cells extracted from the mouse can be cultured at the permissive temperature and the cells will be effectively immortalised. It is then hoped that reverting the cultured cells back to the restrictive temperature will restore differentiation specific gene expression and cell cycle arrest, etc. characteristic of the source tissue. This system should produce more reproducible results on cell lines from the same tissues.
(iii) Double-conditionality on immortalisation:
For greater control than approach (ii) one can transform one""s embryonic stem cells with a construct carrying the conditional immortalisation gene and a conditional promoter. In this way the immortalisation gene is not really expressed in the cells of the organism but cells in culture can be immortalised at the permissive temperature, in the presence of the factor necessary to activate the conditional promoter. This is a variation on approach (ii) but allows the immortalisation to be under tighter control. One example of such a construct carries the SV40 LT tsA58 antigen under the control of the H02Kb Class 1 promoter. This can be used to transform mice to generate the H-2KbtsA58 mouse (A. Kimura et al, Cell 44, 261-272, 1986), which has been used to generate a number of conditionally immortal cell lines. The H-2Kb Class 1 promoter means that the immortalising construct is only expressed at the permissive temperature of the tsA58 protein and is enhanced by exposure of the cells to interferons (B. David-Wattine et al, Immunol. Today 11, 286-292, 1990) Skin fibroblasts prepared from transgenic mice transformed with this construct yielded proliferating cell lines that could be continuously passaged under permissive conditions. The response of these cell lines to interferons varied, presumably as a result of position effects of the integration site of the construct. Other tissues have been isolated from this system as cell lines, and include enteric glia, bone marrow stroma, osteoclast precursors and primitive kidney cells.
According to a first aspect of the present invention there is provided a cell line derived from a transgenic mammal comprising:
(i) a conditional oncogene, transforming gene or immortalising gene or a cell cycle affecting gene; and
(ii) a cell type specific promoter.
Preferably the cell type specific promoter is derived from a secretory tissue.
Preferred cell lines are a neuronal cell line, a mammary cell line, a liver cell line and a kidney cell line.
Preferred cell type specific promoters are the NF-L gene promoter and the MMTV promoter.
Preferably the conditional oncogene, transforming gene, immortalising gene or cell cycle affecting gene is a SV40tsA58 gene, C Erb xcex2 2 gene or TGFxcex1 gene.
The cell cycle affecting gene may however by any gene involved in hyperplasia (excessive formation of cells), cell proliferation or the like.
By utilising a conditional oncogene, transforming gene or immortalisation gene or a cell cycle affecting gene in combination with a cell type specific promoter immortalisation will to some extent be dependant on the differentiation state of the cell. Therefore loss of differentiation specific expression will result in cell-cycle arrest.
A transformation construct of this kind, in which a conditional oncogene, immortalising gene or transforming gene or a cell cycle affecting gene is coupled to a cell type specific promoter, such as the NF-L gene promoter or the MMTV promoter, can be used to generate an immortalised cell line. The conditional oncogene, transforming gene or immortalising gene or a cell cycle affecting gene such as the SV40 tsA58 gene is coupled to a cell specific promoter.
The invention also covers transgenic mammals., particularly rats into whose genomes is integrated at least one recombinant gene whose gene product is involved in oncogenesis, hyperplasia or cell proliferation which may not necessarily be a conditional oncogene like the SV40 tsA58 construct. The invention also applies to transgenic mammals, particularly rats and their progeny who develop hyperplasias or tumours.
The invention also covers the generation of transgenic mammals, particularly rats harbouring tissue specific promoters which drive the expression of cancer causing genes and cell cycle affecting genes, such as ts SV40 T-antigen, c-Erb-B2 and TGF-alpha. The invention also covers cell lines derived from such transgenic mammals, particularly rats.
Transgenic animals are genetically modified animals which harbour at least one experimentally introduced gene. The claimed invention and techniques described are of particular use in generating transgenic animals which can be used as in vivo model systems of normal and diseased tissues, and from which in vitro model systems can be derived. Such model systems are suitable for the study and manipulation of cellular processes in a systematic manner which cannot be achieved with other test systems. Hence they are of great value in elucidating the functions of normal tissue and tissue processes. More importantly they are of value in the validation of candidate drugs and existing drugs, particularly where the model is of a diseased tissue, such as the c-Erb-B2. They are thus of benefit in generating novel therapeutics.
The capability to generate models and cell lines is significant, in that as one moves through the drug development process one can compare the results of in vitro experiments with in vivo experiments more directly.
Toxicological uses of cell lines is also important. Most toxicology is done on whole animals since cell line models of most tissues are unavailable. The conditional immortalisation technique should be amenable to use in other tissues using the relevant tissue specific promoters to generate conditionally immortal cell lines.
The neuronal cell lines developed are of particular significance in that there is no good in vitro model system of normal neuronal tissue in mice, rats or other organisms. Such cell lines will be of benefit for all areas of neuronal research but in particular in drug validation.
Furthermore, having derived an animal from which cell lines can be generated conditionally, one can derive further cell lines by crossing the transgenic animal with animals bearing mutations in other neurone-relevant genes. This will allow researchers to elucidate the roles of such genes and to develop specific in vivo and in vitro disease models.
The benefit derived from the use of the NF-L gene in the transforming construct to generate the neuronal cell lines was unexpected. It is expressed detectably only in post-mitotic neurons so it was surprising that it drove expression of the T-antigen from the SV-40 part of the construct in neuronal precursors. The approach using this construct should be applicable to mammalian systems in general, particularly for the generation of human neuronal cell-lines, particular as a model system in the area of neurodegenerative disease research.
Since mammary tissues are highly secretory-and can be used to secrete protein products, considerable benefit can be derived from developing transgenic animals. A cell line that can be immortalised conditionally is of exceptional value for the generation of transgenic animals or cell lines that secrete particular products either through crossing with other transgenic animals or through direct transgenesis of the animal or derived cell lines.
The approach using for example a MMTV promoter construct should be applicable to mammalian systems in general.
Since current methods for isolating cell lines directly through immortalisation of growing primary cell cultures are inadequate, the applicant has utilised immortalisation of such cells in vivo in transgenic rats Expression of immortalising genes or oncogenes in vivo normally produces tumours and leads to premature death of the animals. However, to allow the mammal, in this case a rat to develop normally the applicant has used an immortalising gene Simian Virus 40 T Antigen gene (or A gene) with a thermolabile mutation of valine for alanine at position 438 (tsA58) (J. Virol 8, 516-524, 1971). The mutated gene is active in the immortalisation process at 33xc2x0 C. but is inactive at 39xc2x0 C. due to the thermal instability of its protein product (Proc. Natl. Acad. Sci. USA. 85, 9076-9080, 1988, Proc. Natl. Acad. Sci. USA. 88, 5096-5100, 1991, J. Cell Sci. 108, 37-49, 1993). Rats are chosen since they are a better model for human systems than the other commonly used laboratory animals (eg. mice), and rats are the animals most widely used in the pharmaceutical industry for drug-screening programmes. This was originally because rats are much larger animals than mice and hence they are far more amenable to the surgical manipulations that many pharmacological studies required. The body temperature of the rat is sufficiently high not to permit this mutated Large T Antigen to be functionally active and to immortalise the cells in vivo (Nature 256, 43-46, 1975).
According to a further aspect of the present invention there is provided a method for producing a transgenic mammal, comprising:
(i) causing a female mammal to super-ovulate;
(ii) mating or artificially inseminating the female mammal;
(iii) obtaining the resulting embryo from the female mammal; and
(iv) incorporating
(i) a conditional oncogene, transforming gene or immortalising gene or a cell affecting gene; and
(ii) a cell specific promoter into the genome of the mammalian embryo.
Preferably the transgenic mammal is a rat and the female rat is made to super-ovulate by supplying her with a regular supply of Follicle Stimulating Hormone (FSH) prior to mating.
By regular is meant more than a twice daily dose, more preferably more than four daily doses, and more preferably still, continuously.
Preferably the female rat is caused to super-ovulate at approximately 30 days of age by continuous infusion with an effective amount of FSH, preferably purified porcine pituitary FSH.
Preferably a female rat is supplied with 2 mg to 8 mg of FSH, more preferably still about 4 mg of FSH, given over approximately one to four, more preferably still, about two days, immediately prior to mating.
The about 4 mg of FSH is preferably given in a saline solution containing from 10 to 30 mg/ml of FSH, more preferably still about 20 mg/ml FSH.
Preferably the FSH used has a low leutinising hormone (LH) to FSH ratio (about 0.10-0.15) such as that produced by Vetrepharm Inc.
The FSH may be injected by any suitable route. Preferably the FSH is injected intraperitoneally.
Preferably the FSH is delivered by a minipump.
According to yet a further aspect of the present invention there is provided a transgenic mammal whose germ cells and somatic cells contain
(i) a conditional oncogene, transforming gene or immortalizing gene or a cell cycle affecting gene; and
(ii) a cell type specific promoter as a result of chromosomal incorporation into the mammalian genome or into the genome of an ancestor of said mammal.
Another aspect of the present invention provides a method for producing a transgenic rat wherein a rat embryo is rinsed substantially free of cumulus cells prior to incorporating an activated oncogene, transforming or immortalizing gene sequence into the genome of the rat embryo.
According to yet a further aspect of the present invention there is provided a method of testing a material suspected of being a carcinogen, said method comprising exposing a mammal produced according to a method of the invention or an ancestor thereof or cells or tissue from a cell line of the invention, to said material and detecting neoplasms as an indication of carcinogenicity.
According to yet a further aspect of the present invention there is provided a method of testing a material suspected of conferring protection against the development of neoplasms, said method comprising treating a mammal produced according to a method of the invention or an ancestor thereof or cells or tissues from a cell line of the invention with said material and detecting a reduced incidence of development of neoplasms, compared to an untreated mammal, as an indication of said protection.
According to yet a further aspect of the present invention there is provided a method of providing a cell line comprising culturing a somatic cell obtained from a transgenic mammal or an ancestor thereof according to the invention.
According to yet a further aspect of the present invention there is provided a cell derived from a cell line obtained from a transgenic mammal or an ancestor thereof according to the invention.
The cells and/or cell lines are preferably tissue specific cell lines, such as, for example, a neuronal In cell line eg NF2C, a mammary cell line eg. B2LT1, a liver cell line, or a kidney cell line. Cell lines NF2C and B2LT1 have been deposited with the ECACC European Collection of Cell Cultures, CAMR (Center for Applied Microbiology and Research), Salisbury, Wiltshire, SP4 OJG, England, as Accession numbers 96092754 and 97032720 respectively.
According to yet a further aspect of the-present invention there is provided a method of providing a transgenic tissue comprising culturing a somatic cell obtained from a transgenic mammal or an ancestor thereof according to the invention.
According to yet a further aspect of the present invention there is provided a tissue derived from a somatic cell obtained from a transgenic mammal or an ancestor thereof according to the invention.
Such a tissue would be of benefit for experimental transplantation and other in vitro work.
Preferably the transgenic rat harbours a tissue specific promoter which drives or controls expression of a cancer causing gene or a cell cycle affecting gene such as tsA58, c-erb B-2 or TGFxcex1. The presence of the promoter may predispose the transgenic rat to cancer, such as breast cancer or to a cell proliferation disorder, for example by over expression of the gene.
Expression of the immortalising gene in the tissue of interest within the transgenic rat is controlled by coupling a tissue-specific promoter that drives the transcription of the immortalising T antigen only within that particular tissue. For example, for targeting expression to the mammary gland the applicant has used the mouse mammary tumour virus (MMTV) promoter (Proc. Natl. Acad. Sci. USA, 82, 5880-5884, 1995) and for targeting expression to neuronal cells the neurofilament light chain (NF-L) promoter (J. Physiol. 84, 50-52, 1990). The former promoter requires activation by hormones released during pregnancy, the latter does not. This targeting technique overcomes the problem encountered previously in transgenic mice which express the tsA58 mutant under the control of a widely-acting constitutive promoter. In that case the mice developed large numbers of tumours in diverse tissues and died at an early age due to revertant mutations in the tsA58 gene (Proc. Natl. Acad. Sci USA 88, 5096-5100, 1991, J. Cell Sci. 104, 37-49, 1993). In the present case cells in the tissue of interest should now grow normally at the restrictive temperature, since they express only the inactive immortalising transgene. However, upon culturing the cells from the tissue of interest at the permissive temperature the immortalising function is activated, and such cells should become immortalised and grow at the permissive temperature (33xc2x0 C.). They should, however, fail to grow and should differentiate successfully at the nonpermissive temperature (39xc2x0 C.).
Production of Transgenic Vectors Carrying the tsA58 Gene
As an example, the thermolabile tsA58 gene was coupled to the mouse MMTV promoter which allows expression in pregnant mammary glands (Proc. Natl. Acad. Sci. USA 82, 5880-5884, 1985) and the rat NF-L promoter which allows expression in CNS neuronal cells of the brain (J. Physiol. 84, 50-52, 1990). Both constructs were introduced into a suitable vector using standard techniques (Molecular Cloningxe2x80x94A Laboratory Manual (2nd edition): Cold Spring Harbour Press, New York, 1989). For example the MMTV promoter was introduced into pUC18 (Gene 3, 103-119, 1985) and the NF-L promoter into pPOLYIII (Gene 57, 193-201,1987). It is not clear whether the whole of the A gene region which codes for both Large T and Small t Antigens is required for immortalisation or just the Large T Antigen region is required. Thus as an example, the tsA58U19 variant of the tsA58 gene was coupled to the MMTV promoter. This combination allows the expression of both Large T and Small t Antigenes, but it fails to replicate because of a mutation, U19, in the region required for replication of the vectorxe2x80x94see FIG. 1A.
FIG. 1A is a plasmid map of the mammary targeting MMTVLTRtsA58U19 construct. A 1.5 kb EcoRI to KpnI fragment of the MMWV LTR containing the start site for transcription, the glucocorticoid response element and the sequences required for mammary specificity, was excised from pMAMneo and ligated into the corresponding restriction endonuclease sites in the cloning vector in pUC18 (Gene 3, 103-119, 1985). Subsequently, the tsA58U19 gene construct containing sequences for both large T antigen and small t antigen, was excised from the BamHI site of pZIPneo (Cell 37, 1053-1062, 1984) and ligated into the BamHI site in pUC18. Restriction endonuclease mapping was carried out to confirm the orientation of the tsA58U19 construct and the identity of the plasmid. The ligation point between the MMTVLTR and tsA58U19 construct was sequenced by the dideoxy chain termination method, to confirm the nature of the ligation point. For the development of transgenic rats, the MMTVLTRtsA58U19 fragment was excised by digestion with EcoRI and SalI, thus minimising the plasmid DNA remaining within the construct.
As the alternative example, the tsA58xcex4t variant of the tsA58 gene was coupled to the NF-L promoter. This combination expresses only Large T Antigen due to a deletion, xcex4t, in the Small t Antigen genexe2x80x94see FIG. 1B.
FIG. 1B is a plasmid map of the neuronal targeting NF-LtsA58xcex4t construct. A 6 kb HindIII fragment of the human neurofilament light chain promoter (J. Physiol. 84 50-52, 1990) was cloned into the HindIII restriction endonuclease site within the polylinker of the cloning vector pPOLYIII (Gene 57, 193-201,1987). The 6 kbp fragment contained the start site for transcription and is sufficient to confer neuronal specific expression. Subsequently a 2.76 kbp KpnI to BamHI fragment of the tsA58xcex4t, which contains a deletion preventing the expression of small t antigen, was cloned into the reciprocal sites within pPOLYIII. The identity of the plasmid was confirmed by restriction endonuclease mapping. The correct nature of the ligation point was confirmed by dideoxy chain termination DNA sequencing. For the development of transgenic rats the NF-LtsA58xcex4t fragment was excised by NotI restriction endonuclease digestion, minimising the amount of plasmid DNA within the construct.
The excised gene constructs were fractionated by 0.8% agarose gel electrophoresis and the requisite DNA band excised. The DNA was not exposed to ethidium bromide or UV light directly, but a separate marker lane of DNA was run, and used to identify the region of the non-stained gel containing the required fragment. The DNA fragment was then extracted from the agarose slice using the Geneclean Bio 101 kit (2.1.5) with the final pellet being resuspended in a low salt buffer (0.2M NaCl, 20 mM Tris pH7.4, 1.0 mM EDTA). The resulting DNA was further purified using the ELUTIP-d kit (Schleicher and Schuell). Briefly, the ELUTIP-d ionic exchange column was washed with a high salt buffer (1.0 M NaCl, 20 mM Tris HCl pH7.4, 1.0 mM EDTA) and then primed with a low salt buffer (0.2M NaCl, 20 mM Tris HCl pH7.4, 1.0 mM EDTA). The DNA suspension was then run through the column such that the DNA attached to the matrix. Finally the DNA was eluted with high salt solution (1.0 M NaCl, 20 mM Tris HCl pH7.4, 1.0 mM EDTA). The resulting DNA was then ethanol precipitated (2.1) and resuspended in injection buffer (10 mM Tris HCl, 0.1 mM EDTA, pH7.4) at a concentration of 1.5 xcexcg/ml.
Production of Transgenic Rats
Transgenic Sprague Dawley rats were produced by standard methods of microinjection of DNA into the pronuclei of single-cell rat embryos as for transgenic mice (Manipulating the Mouse Embryo. Cold Spring Harbor Press, New York, 1986), except where indicated below. The stud male was mated with a super-ovulating female and the embryos were collected from the oviduct of the female. The transgene in the vector was microinjected into a single pronucleus of a fertilised embryo, which was then introduced into the oviduct of a pseudo-pregnant foster-mother.
The major difference in the technique over that used in transgenic mice was that the female donor rats were found to require a regular/continuous supply of Follicle Stimulating Hormone (FSH) for successful super-ovulation and this was most suitably delivered by a minipump. Without this regular/continuous supply of FSH the injected rat embryos subsequently failed to develop in their foster-mothers. Thus Sprague-Dawley female rats from Charles Rivers Labs, UK were superovulated at 30 days of age by regular/continuous infusion of purified porcine pituitary follicle-stimulating hormone FSH; equivalent to the National Institute of Health (USA) reference standard (NIR-FSH-P1). (Vetrepharm Inc., London, Ontario)) via Alzet mini-osmotic pumps (Alzet model 2001; Alza Scientific Products, Palo Alto, Calif.) (Biol. Reprod. 39, 511-518, 1988). Each pump was filled with 200 xcexcl of FSH diluted to 20 mg/ml in sterile isotonic saline. Pumps were inserted intraperitoneally into pentobarbital-anaesthetized animals, two days prior to mating to deliver 2 mg continuously per 24 hours. Synchronisation of ovulation was induced 48-52 hours later by an intraperitoneal injection of 100 ng luteinising hormone releasing hormone analogue [des-gly10 (D-ala)-LHRH-ethylamide, Sigma]. After mating the females overnight with males of proven fertility and examining for vaginal plugs, all females were sacrificed by cervical dislocation. The pumps were transferred to a second set of animals, and embryos were collected in Dulbecco""s PBS from the oviducts (swollen ampullae) of plugged females.
Another difference was that more skill of microinjection of the pronucleus was required over that of the mouse, because of less optical resolution of the pronucleus of the rat compared with that of the mouse. Thus embryos were rinsed free of cumulus cells in 0.1% hyaluronidase and transferred to modified M2 medium for microinjection or modified M16 medium (280 mOsm) for culture at 38.5xc2x0 C. in 5% CO2 until pronuclei became distinguishable (Manipulating the Mouse Embryo. Cold Spring Harbor Press, New York, 1986). Pronuclear injections were performed on a Nikon inverted microscope equipped with Narishige micromanipulators and Normarski optics. The DNA was dissolved in 1xc3x97 injection buffer (10 mM Tris HCl, 0.1 mM EDTA, pH7.4) and injected at a concentration of approximately 2 ng/xcexcl (Proc. Natl. Acad Sci. USA, 82, 4438-4442, 1985). After injection of one pronucleus in each embryo (as evidenced by pronuclear expansion), all embryos were transferred to modified M16 medium for incubation until oviduct transfers could be performed.
Transfers of injected embryos into pseudopregnant foster mothers were performed under a standard dissection microscope with a Nikon cold light source. After a solution of 0.1% epinephrine was applied to the ovarian bursa to inhibit bleeding, the bursa was torn to allow access, and the embryos were transferred to the two horns of the oviduct using a finely drawn glass pipette. From experience only bilateral transfers were performed. In addition, embryos were transferred at the pronuclear stage into Day 1-pseudopregnant recipients (synchronous) or, after overnight culture, at the early 2-cell stage into Day 1 (asynchronous) or Day 2 (synchronous) recipients.
These techniques ensured for the first time that transgenic rats could be developed routinely. Further examples of the use of these techniques that the applicants have employed are the introduction of different oncogenes into the pronucleus of the rat embryo and their successful incorporation and expression in transgenic offspring are given later. In all cases after successful births and rearing of the animals, they were mated together to produce the F1 generation.
Animals Bearing the Mammary-targeting Construct
The MMTVLTRtsA58U19 construct contains a 1.5 kbp fragment of the MMTV Long Terminal Repeat (LTR), which contains the elements for glucocorticoid-specific induction, specificity for expression and the start site for transcription, and this was cloned upstream of tsA58U19 (FIG. 1A). After the vector containing this construct was microinjected into embryos, and they were reimplanted and reared in foster-mothers, one founder that contained the transgene was identified out of 43 rats tested. The rats were tested by hybridising the 5xe2x80x2 region of the tsA58 gene to DNA isolated from their tails. This represents a success rate of 2.3%. The copy number of transgenes in the founder was two. The F1 progeny produced from the founder rat demonstrated Mendelian inheritance and both hemizygotes and homozygotes have been identifiedxe2x80x94see Table 1 below.
The animals suffered no apparent side effects due to the transgene.
Expression of the transgene has been detected by immunocytochemistry using an antibody to Large T Antigen (J. Virol. 39, 861-869, 1981) but only in growing and lactating mammary glandsxe2x80x94see FIGS. 2A and 2B and hardarian gland, but in no other organs from lactating ratsxe2x80x94see Table 2 below:
FIG. 2A and 2B are immunocytochemical stainings for large T Antigen in the mammary glands of transgenic rats. Mammary glands from mammary-targeted MMTVLRtsA58U19 transgeneic rats incubated with anti-Large T Antigen (Table 2) showing staining in (FIG. 2A) growing mammary glands and (FIG. 2B) lactating mammary glands, 6 days post partum. Magnificationxc3x97220; Bar=50 xcexcm.
Animals Bearing the Brain-targeting Construct
The NF-LtsA58xcex4t construct contains a 6 kbp fragment of the human neurofilament light chain promoter, which, itself contains the elements required for specific expression and also the start site for transcription, and this was cloned upstream of tsA58dt. After the embryos were microinjected with this vector and reared in foster-mothers, one founder was identified from 15 rats screened, a success rate of 6.7%. The founder was shown to be mosaic, as the resultant progeny did not inherit the transgene in a Mendelian mannerxe2x80x94see Table 3 below.
The copy number of the transgene in the F1 was approximately 6. At present only hemizygous animals have been identified. Subsequent breeding of the hemizygous animals has exhibited Medelian inheritance (Table 3).
Expression of the transgene has been identified by immunocytochemistry, using antibody to Large T Antigen, in various regions of the adult brain, but in no other organs (Table 2). The most intense staining was found in the fibres of the internal capsule which are the major neuronal highway between the thalamus and the cortexxe2x80x94see FIG. 2C. Staining was also noticed in the choroid plexus which is involved in the production of cerebro-spinal fluidxe2x80x94see FIG. 2D.
FIGS. 2C and 2D illustrate immunocytochemical staining for Large T Antigen in the brains of transgenic rats. Brain from neuronal-targeted NF-LtsA58xcex4t transgenic rats incubated with anti-Large T Antigen above showing staining in (FIG. 2C) fibres of the internal capsule and (FIG. 2D) choroid plexus. Magnificationxc3x97220: Bar=50 xcexcm.
The NF-LtsA58xcex4t transgenic rats suffer side effects potentially due to the transgene in later life. Three animals developed choroid plexus tumours which showed high levels of staining for Large T Antigen. The development of tumours may be due to the reversion of the transgene to that of the wild-type. A number of animals have also died due to renal failure, but the expression of the transgene could not be detected in this organ.
Production of Cell Lines from Mammary-targeted Tissue
Primary cultures were established from the mammary glands of 50-day old virgin female rats that express the mammary-targeted T Antigens by digestion with collagenase (J. Cell Biol. 73, 561-577, 1977). Fibroblasts were removed by preplating or by centrifugation through percoll gradients (In Vitro 22, 429-439, 1986). The primary cultures were grown in 50% DMEM, 50% RPMI, 10% FCS, 20 ng/ml EGF, 50 ng/ml hydrocortisone, 50 ng/ml insulin and medium exposed to UV-irradiated Rama 27 feeder fibroblasts (In Vitro, 25, 23-36, 1989). The primary cultures were transferred by treatment with EDTA (Cell 15, 283-298, 1978). After 4 passages, an epithelial-like cell strain was establishedxe2x80x94see FIG. 3A.
FIGS. 3A-3D show phase-contrast macrographs of all cell lines produced from transgenic rats at a magnification of X200, Bar=50 xcexcm.
FIGS. 3A and 3B are a B2LT1 cell line from the mammary glands of mammary-targeted MMTVLTRtsA58U19 transgenic rats.
In FIG. 3A the cell line was kept at 33xc2x0 C. and shows an epithelial-like intermediate morphology, and in FIG. 3B the cell line was kept at 39xc2x0 C. with 5 ng/ml prolactin and shows a dark droplet cuboidal morphology with associated hemispherical blisters or domes reminiscent of cultured alveolar cells.
FIGS. 3C and 3D are a NF2C cell line from the brain of neuronal-targeted NF-LtsA58xcex4t transgenic rats.
In FIG. 3C the cell line was kept at 33xc2x0 C. and shows more-elongated cells, and in FIG. 3D the cell line was kept at 39xc2x0 C. with 5 ng/ml basic fibroblast growth factor and shows dendritic-like outgrowths.
This epithelial like cell strain from the mammary glands was cloned and stored in frozen aliquots. The cloned cell line B2LT1 has been characterised by immunofluorescence, and it stains for milk fat globule membrane, cytokeratins and peanut lectin, as well for Large T Antigen (J. Virol 39, 861-869, 1981)xe2x80x94see Table 4 below.
It therefore exhibits some properties of epithelial cells (J. Histochem. Cytochem. 37 1087-1100, 1989). However, the epithelial-like cells convert to a more-elongated, anti-vimentin, pokeweed mitogen (J. Histochem Cytochem. 38, 1633-1645; 1990) and weakly anti-actin staining myoepithelial-like phenotype (J. Histochem. Cytochem. 37, 1087-1100, 1989) in culture. This result is consistent with the original epithelial cells being mammary epithelial stem cells (Histol. Histopathol. 8, 385-404, 1993). When they were grown at the restrictive temperature for the transgene in the presence of prolactin, B2LT1 cultures ceased to dividexe2x80x94see FIG. 4A and took on the appearance of alveolar cells in culture (FIG. 3B) (Eur. J. Biochem. 133, 707-715, 1993) and showed enhanced staining for epithelial/alveolar cells and reduced staining for myoepithelial-like cellsxe2x80x94see Table 4 above.
FIG. 4A shows the growth curves for mammary derived B2LT1 cells. The B2LT1 cells were seeded at a density of 6xc3x97104 cells per plate and incubated at 33xc2x0 C. for 24 hours after which time half of the cells were transferred to an incubator at 39xc2x0 C. Cell numbers were counted for quadruplicate replica plates, at two day intervals using a Coulter counter. The meansxc2x1SE are shown.
The level of chemiluminescent staining for the neurone-specific markers was assessed by densitometry of the radioautographs and comparison with the level for 10 xcexcg of rat brain extract which was arbitarily set at 100%. The levels of Chemiluminescent staining were recorded as follows: ++++. 75-100%; +++, 50-75%; ++, 25-50%; +, 5-25%; and xe2x88x92,  less than 5% of the level of rat brain extract. The level of chemiluminescent staining of MAb pAB 419 to the N-terminus of Large T Antigen (J. Virol 39, 861-869, 1981) was assessed relative to that of 10 xcexcg of protein of the Huma 62 SV40-immortalised cell line (Dec. Bio. 136, 167-180, 1989) arbitarily set at 100%.
The B2LT1 cells have now lost the requirement for feeder cells in culture and grow rapidly at 33xc2x0 C. with a doubling time of 22 hr. The cells grow to a density of 4xc3x97106 per 9 cm diameter plate and when split at a ratio of 1:10 are confluent after 3 days at 33xc2x0 C.
Production of Cell Lines from Tissue Containing the Brain-targeted Transgene
Cell lines were developed by establishing primary cultures of brain cells of an adult male by digestion with trypsin and culturing them at 33xc2x0 C. in 50% DMEM, 50% RPMI, 10% FCS with added pyruvate, glutamine, NaHCO3 and 50 ng/ml insulin (Dev. Neurosci. 5, 2197-2200, 1994). The cells were transferred as above. At crisis, the more abundant glial and fibroblast cells died out allowing the slower-proliferating neuronal cells to be cultured. These were eventually cloned, one such clone was designated NF2C, and it was cultured and stored in aliquots at xe2x88x9270xc2x0 C.
The cells grow successfully at 33xc2x0 C. in 50% DMEM, 50% RPMI, 10% FCS and 50 ng/ml insulin (In Vitro, 25, 23-36, 1989), with a doubling time of 36 hr and grow to a density of approximately 2xc3x97106 per 9 cm diameter plate. At the restrictive temperature of 39xc2x0 C. the cells ceased to dividexe2x80x94FIG. 4B
FIG. 4B shows the growth curves for brain-derived NF2C Cells. The NF2C cells were seeded at a density of 2xc3x97105 cells per plate and incubated at 33xc2x0 C. for two days, such that the cells may become established after passaging. After 2 days, half of the plates were transferred to an incubator at 39xc2x0 C. Cell numbers were counted for quadruplicate plates at three day intervals using a Coulter counter. The meansxc2x1SE are shown.
At 33xc2x0 C. the cells are elongated (FIG. 3C) which becomes further exaggerated at 39xc2x0 C. (FIG. 3D). The cell line has been characterised by Western blotting of cellular extracts with suitable antibodies for neuronal markersxe2x80x94see FIG. 5.
FIG. 5 shows immunochemical detection of neuronal markers in brain derived cells in NF2C. Proteins (10 xcexcg) (A) from homogenised brains of normal rats; (B) from a rat mammary-epithelial cell line Rama 37 (Cancer less than etastasos Rev. 6, 55-83, 1987); (C,D) from the brain-derived and neuronal-targeted NF-LtsA58xcex4t cell line NF2C grown at (C) 33xc2x0 C. or (D) 39xc2x0 C. with 5 ng/ml basic fibroblast growth factor were electrophoresed on 12.5% sodium dodecylsulphate polyacrylamide gels and subject to Western blotting (Molecular cloningxe2x80x94A Laboratory Manual (2nd Edition): Cold Spring Harbour Press, New York, 1989) with antibodies to markers of neuronal cells: neuron-specific enolase (Ann. Rev.Neurosci. 10, 269-295, 1987), synaptophysin (Cell 41, 1017-1028, 1985), MAP2 (Ann. Rev. Neurosci. 11, 29-44, 1988), tau (J. Cell Biol. 102, 252-262, 1986), neuraofilament protein (Proc. Natl. Acad. Sci. USA., 79, 1326-1330, 1982) and to a marker of glial cells, GFAP (J. Cell Biol. 88, 115-126. 1982). The antibody-bound proteins were visualised by the ABC method (J. Histochem. Cytochem. 29, 577-601, 1981) using chemiluminescence (Clin. Chem. 25, 1531-1546, 1979) as for Table 5. The chemiluminescence was photographed using a MacIntosh computer video capture, and the Image 1.44 VDM-F photo programme. Molecular weight marker proteins were run in parallel and the molecular weights of the stained bands were recorded.
The cells express neuron-specific enolase (Ann. Rev. Neurosci. 10, 269-295, 1987), synaptophysin (Cell 41, 1017-1028, 1985), microtubule-associated protein 2 (MAP2) (Ann. Rev. Neurosci. 11, 29-44, 1988), tau (J. Cell Biol. 102, 252-262, 1986) and neurofilament protein (Proc. Natl, Acad. Sci. USA. 79, 1326-1330, 1982) strongly at 39xc2x0 C. and more weakly or not at all at 33xc2x0 C. The glial marker 1 glial fibrillary acidic protein (GFAP) (J. Cell Biol. 88 115-126, 1982) was expressed weakly at 33xc2x0 C. but not at 39xc2x0 C., and Large T Antigen was identified at both temperatures (Table 5). Expression of the neuronal-specific markers and dendritic outgrowths was increased if the cells were grown in medium containing basic fibroblast growth factor which acts like a short-acting nerve growth factor for 6-7 days in these systems (J. Neurosci 5, 307-316, 1985). These results suggest that the cell line from the brain is of neuronal origin and that it may possess certain stem cell characteristics being capable of expressing glial markers as well as neuronal markers but only at the permissive temperature.